Immunoassays

Immunoassays are based on the principles that specific antigens will stimulate very specific and unique immune responses and that the proteins produced by the immune response, called antibodies, can be used to signal the presence of a target compound in a sample. The principle of an indirect EIA for antibody is when an antibody in the specimen is bound by a "capture antigen" which has been attached to a solid surface. The bound antibody is detected by binding an added enzyme conjugated anti- human antibody. The bound enzyme labeled antibody is detected by its ability to break down its substrate to a coloured product. Although enzyme immunoassays can be set up in several different formats the indirect method is the most appropriate for the detection of total specific antibody.

TLC

Chromatography is a method of separating mixtures of two or more compounds. The separation is accomplished by the distribution of the mixture between two phases: one that is stationary and one that is moving. Chromatography works on the principle that different compounds will have different solubilities and adsorption to the two phases between which they are to be partitioned.

Thin Layer Chromatography (TLC) is a solid-liquid technique in which the two phases are a solid (stationary phase) and a liquid (moving phase). Solids most commonly used in chromatography are silica gel (SiO2 x H2O) and alumina (Al2O3 x H2O). Both of these adsorbents are polar, but alumina is more so. Silica is also acidic. Alumina is available in neutral, basic, or acidic forms.

TLC involves spotting the sample to be analyzed near one end of a sheet of glass or plastic that is coated with a thin layer of an adsorbent. The sheet, which can be the size of a microscope slide, is placed on end in a covered jar containing a shallow layer of solvent. As the solvent rises by capillary action up through the adsorbent, differential partitioning occurs between the components of the mixture dissolved in the solvent the stationary adsorbent phase. The more strongly a given component of a mixture is adsorbed onto the stationary phase, the less time it will spend in the mobile phase and the more slowly it will migrate up the plate.

Rapid Methods

The rapid detection of pathogens and other microbial contaminants in food is critical for ensuring the safety of consumers. Traditional methods to detect foodborne bacteria often rely on time-consuming growth in culture media, followed by isolation, biochemical identification, and sometimes serology. Recent advances in technology make detection and identification faster, more convenient, more sensitive, and more specific than conventional assays -- at least in theory. These new methods are often referred to as "rapid methods", a subjective term used loosely to describe a vast array of tests that includes miniaturized biochemical kits, antibody- and DNA-based tests, and assays that are modifications of conventional tests to speed up analysis. Some of these assays have also been automated to reduce hands-on manipulations. With few exceptions, almost all assays used to detect specific pathogens in foods require some growth in an enrichment medium before analysis.

There are many DNA-based assay formats, but only probes, PCR and bacteriophage have been developed commercially for detecting foodborne pathogens. Probe assays generally target ribosomal RNA (rRNA), taking advantage of the fact that the higher copy number of bacterial rRNA provides a naturally amplified target and affords greater assay sensitivity.

The basic principle of DNA hybridization is also being utilized in other technologies, such as the polymerase chain reaction (PCR) assay, where short fragments of DNA (probes) or primers are hybridized to a specific sequence or template, which is then enzymatically amplified by Taq polymerase using a thermocycler. Theoretically, PCR can amplify a single copy of DNA by a million fold in less than 2 hrs; hence its potential to eliminate, or greatly reduce the need for cultural enrichment. However, the presence of inhibitors in foods and in many culture media can prevent primer binding and diminish amplification efficiency, so that the extreme sensitivity achievable by PCR with pure cultures is often reduced when testing foods. Therefore, some cultural enrichment is still required prior to analysis.

The enzyme-linked immunosorbent assay (ELISA) is the most prevalent antibody assay format used for pathogen detection in foods. Usually designed as a "sandwich" assay, an antibody bound to a solid matrix is used to capture the antigen from enrichment cultures and a second antibody conjugated to an enzyme is used for detection. The walls of wells in microtiter plates are the most commonly used solid support; but ELISAs have also been designed using dipsticks, paddles, membranes, pipet tips or other solid matrices.

Applications and Limitations of Rapid Methods
Almost all rapid methods are designed to detect a single target, which makes them ideal for use in quality control programs to quickly screen large numbers of food samples for the presence of a particular pathogen or toxin. A positive result by a rapid method however, is only regarded as presumptive and must be confirmed by standard methods. Although confirmation may extend analysis by several days, this may not be an imposing limitation, as negative results are most often encountered in food analysis. Most rapid methods can be done in a few minutes to a few hours, so they are more rapid than traditional methods. But, in food analysis, rapid methods still lack sufficient sensitivity and specificity for direct testing; hence, foods still need to be culture-enriched before analysis. Although enrichment is a limitation in terms of assay speed, it provides essential benefits, such as diluting the effects of inhibitors, allowing the differentiation of viable from non-viable cells and allowing for repair of cell stress or injury that may have resulted during food processing.

Electroimmunoessays

Electroimmunoassays consists of a circuit with a capture antibody which is attached to the solid surface of the electrode gap area. This technology to isolate and identify foodborne pathogens combines specific antibody-antigen binding to the production of an electrical signal. The target antigen binds to the capture antibody when the sample is added. Following that, a colloidal gold–labeled detection antibody is bound to create a capture-target-detector sandwich. Finally, the silver ions are deposited onto the colloidal gold, which produces a conductive silver bridge hence closing the circuit and resulting in a measured drop in resistance.

DNA Probe

The identification of bacteria by DNA probe hybridization methods is based on the presence or absence of particular genes. This is in contrast to most biochemical and immunological tests that are based on the detection of gene products such as antigens or chemical end products of a metabolic pathway. A DNA probe is composed of nucleic acid molecules, most often double-stranded DNA. It consists of either an entire gene or a fragment of a gene with a known function. Alternatively, short pieces of single-stranded DNA can be synthesized, based on the nucleotide sequence of the known gene. These are commonly referred to as oligonucleotides. Both natural and synthetic oligonucleotides are used to detect complementary DNA or RNA targets in samples. Double-stranded DNA probes must be denatured before the hybridization reaction; oligonucleotide and RNA probes, which are single-stranded, do not need to be denatured. Target nucleic acids are denatured by high temperature or high pH, and then the labeled gene probe is added. If the target nucleic acid in the sample contains the same nucleotide sequence as that of the gene probe, the probe will form hydrogen bonds with the target. Thus the labeled probe becomes specifically associated with the target. The unreacted, labeled probe is removed by washing the solid support, and the presence of probe-target complexes is signaled by the bound label.

The physical basis for gene probe tests stems from the structure of DNA molecules themselves. Usually, DNA is composed of two strands of nucleotide polymers wound around each other to form a double helix. These long nucleotide chains are held together by hydrogen bonds between specific pairs of nucleotides. The hydrogen bonds holding the strands together can usually be broken by raising the pH above 12 or the temperature above 95°C. Single-stranded molecules result and the DNA is considered denatured. The source of the DNA strands is inconsequential as long as the strands are complementary. If the strands of the double helix are from different sources, the molecules are called hybrids and the process is termed hybridization.

Laboratory Methods

DNA and Protein-based methods have been developed and applied for the detection of GMO. For the detection of Genetically Modified Organisms (GMOs) at the level of DNA, PCR based methods are mainly used, whereas for protein-based detection, immunoassays such as Western blot, ELISA, and lateral strips methods are predominantly used.

DNA -based methods are primarily based on multiplying a specific DNA with the PCR technique. The PCR reaction allows the million or billion fold amplification of a specific target DNA fragment framed by two synthetic oligonucleotide primers. The PCR is done by a multiple-process with consecutive cycles of denaturation step to denature DNA, annealing step where primers find their correspondent complementary sequences on the template and extension or elongation step where Taq polymerase copies the complementary strand. In each cycle three different steps were progressed in different temperatures. To perform the reliable PCR test, it is important that specific primers were designed to bind selectively to the complementary sequences of the target DNA; the specific gene introduced, promoter and terminator, a sequence spanning between a regulatory sequence and a sequence between the gene and the flanking genome A prerequisite for the PCR-based technique is to know specific DNA sequence inserted in GMO, served as target and to find adequate standard materials to be used as positive analytical controls. Three kinds of PCR strategies are currently used for GMO detection; multiplex PCR, quantitative competitive PCR (QC-PCR) and real-time PCR.

Immunoassay is based on the specific binding between an antigen and an antibody. Thus, the availability of antibodies with the desired affinity and specificity is the most important factor for setting up immunoassay systems. This technology is ideal for qualitative and quantitative detection of many types of proteins, however can’t be applied in highly processed soy products

ELISA is an enzyme linked immuosorbant assay. This technique uses an antibody that recognizes specific proteins. Protein is bound to a well of a plastic plate instead of a membrane. Antigen and antibody bind and produce a stable complex, which can be visualized by addition of a second antibody linked to an enzyme. The advantages of ELISA are to be capable of quantitative analysis when a standard curve is included, and high throughput.

Main Issues of Concern for Human Health

The three main issues debated are tendencies to provoke allergic reaction (allergenicity), gene transfer and outcrossing.

Allergenicity
As a matter of principle, the transfer of genes from commonly allergenic foods is discouraged unless it can be demonstrated that the protein product of the transferred gene is not allergenic. While traditionally developed foods are not generally tested for allergenicity, protocols for tests for GM foods have been evaluated by the Food and Agriculture Organization of the United Nations (FAO) and WHO. No allergic effects have been found relative to GM foods currently on the market.

Gene transfer
Gene transfer from GM foods to cells of the body or to bacteria in the gastrointestinal tract would cause concern if the transferred genetic material adversely affects human health. This would be particularly relevant if antibiotic resistance genes, used in creating GMOs, were to be transferred. Although the probability of transfer is low, the use of technology without antibiotic resistance genes has been encouraged by a recent FAO/WHO expert panel.

Outcrossing
The movement of genes from GM plants into conventional crops or related species in the wild (referred to as “outcrossing”), as well as the mixing of crops derived from conventional seeds with those grown using GM crops, may have an indirect effect on food safety and food security. This risk is real, as was shown when traces of a maize type which was only approved for feed use appeared in maize products for human consumption in the United States of America. Several countries have adopted strategies to reduce mixing, including a clear separation of the fields within which GM crops and conventional crops are grown.

Potential Risks to Human Health

• direct health effects (toxicity)
• tendencies to provoke allergic reaction (allergenicity)
• specific components thought to have nutritional or toxic properties
• the stability of the inserted gene
• nutritional effects associated with genetic modification
• any unintended effects which could result from the gene insertion

Positive Environmental Issues

1. Reduced Pesticide Usage
   Reduced pesticide usage is one of the benefits of genetically modified crops that are pest resistant. Currently, genetically modified pest resistant crops include Bt cotton, Bt corn, Bt sweet corn, Bt potatoes, and virus resistant squash. These crops are able to resist certain pests and need fewer pesticide sprays. In the past, pesticide usage on cotton, sweet corn and potatoes has been very high with some of these crops requiring more than a dozen insecticide sprays per season. However, Bt sweet corn needs less than 15 percent of the insecticide sprays than those traditional varieties.But Bt crops still do need some insecticide sprays. Bt is very selective and only controls some insects, and the protection provided by the Bt only protects against some pests. So while Bt crops are protected from the primary pests, control of secondary pests may sometimes require the use of insecticide sprays.
   
2. GM Crops Compliment Biological Control
   One group of non-target organisms that need to be encouraged is the natural enemies of our crop pests. Natural enemies are composed of a wide array of parasitic and predatory insects and other arthropods. Control of crop pests by natural enemies is referred to as biological control. Unfortunately, biological control cannot prevent crop damage in all circumstances and farmers often need to apply pesticide sprays.When these sprays include non-selective insecticides, the natural enemy populations are often hurt more than the pest that needed controlling. The reason is that while the pesticide may kill both the pest and its natural enemies, by killing the pest it has also eliminated the food source that the natural enemy populations will need to recover. Because of this, it often takes much longer for the natural enemy populations to recover than the pest population. In the absence of natural enemies, pest populations are able to increase much more rapidly. This can result in greater reliance on pesticide sprays after the natural enemies are eliminated.Genetically modified crops that produce their own plant pesticides are more compatible with biological control. The plant pesticides are more selective than most insecticide sprays.In addition, because the need fewer pesticide applications, they preserve natural enemies populations and are more compatible with biological control.
   
3. Plant Pesticides Impact less on Non-target Organisms
   Genetically modified plants that produce their own plant pesticides include Bt cotton, Bt corn, Bt sweet corn, and Bt potatoes. These plant pesticides are very selective, for example, the type of Bt in Bt corn only controls the caterpillars of some moths and butterflies. The type of Bt in Bt potatoes controls Colorado potato beetles. In addition, the Bt is inside the plant, so only insects that feed on the plant or plant parts are exposed to the plant pesticide. An exception to this is with the pollen from Bt corn which is wind blown. The Bt-corn pollen also contains the Bt toxin. It has been shown in the laboratory to reduce the survival of monarch caterpillars that have been feed on milkweed plants that were dusted with this pollen.But it is important to keep in mind that these genetically modified crops that produce their own plant pesticides require fewer pesticide sprays. Most of the commonly used insecticides used on these crops are referred to as broad spectrum insecticides. They are generally as toxic to non-target organisms as they are to the target pest. Plants that produce their own plant pesticides are more selective in terms of controlling pests without damaging non-target organisms. Their impact on non-target organisms is further reduced because they require fewer broad spectrum pesticide sprays.
   
4. Increased Yields, Reducing the Need to Expand Agricultural Acreage
   While the genetically modified crops on the market today do not increase yields. For example, the GM crops that produce their own plant pesticides do not yield more than traditional varieties; they just protect the plants from yield loss. Differences in yield do not represent the ability of the plant to produce more. In fact, in the absence of pests, these hybrids should have yields equal to comparable to traditional hybrids.However, GM crops that increase yields are under development and the future looks very promising. Unless yield increases are able to keep in pace with population growth, more land will be need to be devoted to commercial agriculture. Current tends show that the amount of prime agricultural land available is decreasing. Crop yields may need to increase by 20 to 40 percent in the next 20 years in order feed an expanding population. Biotechnology provides some of the tools needed to continue to increase the yields of the world's important staple crops.
5. Issue: Some GM Crops May Reduce Soil Erosion
   New herbicide resistant crops may help to reduce soil erosion. There is a need to prevent soil erosion in order to maintain farm sustainability and to reduce pollution of streams, rivers and wetlands. This allows the producer greater flexibility in terms of when to control weeds. Rather than using reemergence herbicides that may need to incorporated into the soil, these are applied over the crop and the weeds as they are actively growing. GM herbicide resistant crops are compatible with and encourage no-till agriculture.
   

Controversies

Controversies surrounding GM foods and crops commonly focus on human and environmental safety, labelling and consumer choice, intellectual property rights, ethics, food security, poverty reduction, and environmental conservation.

Safety
Potential human health impact: allergens, transfer of antibiotic resistance markers, unknown effects Potential environmental impact: unintended transfer of transgenes through cross-pollination, unknown effects on other organisms (e.g., soil microbes), and loss of flora and fauna biodiversity

Labelling
-Not mandatory in some countries (e.g., United States)
-Mixing GM crops with non-GM confounds labelling attempts

Access and Intellectual Property
-Domination of world food production by a few companies
-Increasing dependence on Industrialized nations by developing countries
-Bio-piracy—foreign exploitation of natural resources

Ethics
Violation of natural organisms' intrinsic values
Tampering with nature by mixing genes among species
Objections to consuming animal genes in plants and vice versa
Stress for animal

Society
New advances may be skewed to interests of rich countries

Genetically Modified Food

Genetically Modified (GM) foods are produced from genetically modified organisms (GMO) which have had their genome altered through genetic engineering techniques. The general principle of producing a GMO is to insert DNA that has been taken from another organism and modified in the laboratory into an organism's genome to produce both new and useful traits or phenotypes. Typically this is done using DNA from certain types of bacteria. Genetic engineering techniques allow scientists to insert specific genes into a plant or animal without having to go through the trial-and-error process of selective breeding. Therefore it is extremely rapid compared to selective breeding.

Product recall

Product Recall Plan includes:

1. Recall Management Team

Include members from different departments of the firm

• production-line workers
• quality control
• purchasing and marketing
• sales
• legal services
• accounting
• technical
• distribution
• public relation departments

Recall team’s task:

• to evaluate whether the product constitutes a threat to the health or safety of the consumer
• recommend whether a recall should be initiated and the appropriate recall strategy

2. Recall Contact List

• allows effective and fast delivery of notices to put unsafe products off shelves as quickly as possible
• contact list includes:
• recall team
• senior management
• suppliers of all ingredients
• distributors
• sources of technical advice and support (like laboratory facilities)
• local regulatory authorities (Food Control Department; USA regulatory authorities: USDA and FDA).

3. Product Recall Decision Tree

• to ensure a systematic and logical approach at whether to execute recall plan or not
• different strategies for different classes of recall as follows:
  

a) Class 1 Recall: Carried out when there is a reasonable probability that the use of product
will cause serious adverse health consequences or death

b) Class 2 Recall: Carried out when defect products may cause temporary or medically
reversible adverse health consequences and the probability of serious
adverse health consequence is remote.
c) Class 3 Recall: Carried out when defect products is not likely to cause health
consequences, but violates some specific food regulation.

4. Scope of Recall

• to decide which batch of products should be recalled

5. Records

• determine the causes of adulteration and help in defining the scope of recalls

6. Products’ Traceability

• allows identification of unsafe products
• limits the scope of the recall
• removes the products from sale quickly and accurately
• products can be identified accurately with product names, product description and batch codes

7. Recall Procedures

• a set of procedures on how products are to be identified, collected, disposed off and post-recall follow-up during a recall
• include how communications are made between food manufacturers, distributors and consumers

8. Records of Recalled Products

• to ensure that the quantity of unsafe products distributed tallies with the quantities of recalled products
• serve as a reference and would be needed to show to the local regulatory (Food Control Department) during recall follow-up

9. Testing and Reviewing of Product Recall Plan
The plan should be examined for errors, particularly in the contact lists or in light of any changes in the company’s product recall or trading status. It is essential that a product recall plan is periodically tested using a ‘trial run’ or mock recall exercise. This can be considered as a validation of the product recall plan. This procedure should also be documented and held as part of the product recall plan itself. Companies that develop product recall plans but do not test them may face problems when a real food safety incident occurs. Food businesses involved in product recall should review the product recall process and amend the product recall plan if necessary.

Food Safety

What is Food Safety?

Food safety is a scientific discipline describing the handling, preparation and storage of food in ways to prevent food borne illness. Randomly sampling of product biologically, physically and chemically is done to ensure that that batch of production is safe for consumption, within legal issues and of good quality.

Food can potentially become contaminated through improper practices from the time it is produced to the time it is consumed. Hence, from the government bodies to the people in the food industries to us consumers, everyone plays an important role in maintaining food safety.

Who ensures food safety?

The government is responsible for establishing a framework that promotes the delivery of safe food by the industry, and the provision of adequate information to consumers.The Agri-Food and Veterinary Authority (AVA) is the national authority for food safety in Singapore. To ensure that food sold in Singapore is safe, AVA sets stringent food safety standards that are consistent with international standards.

People in the food industry have to maintain high food safety standards by complying with AVA's stringent requirements.

Datails of vegetables

Vegetables – Diseases and Possible Microbes which can cause illness.


Food poisonings – caused by Bacillus cereus


Botulism Type A and B – caused by Clostridium botulinum


Listerosis – caused by Listeria monocytogenes


E.coli infection – caused by Escherichia coli


Ascariasis – caused by Ascaris lumbricoids

Choices we made

The question of which food product we choose still lingers...choises can be both a blessing and a curse..

My group consists of memebers from various SIP companies, ranging from Mcdonald to KFC to Sakae Sushi to Burger King. Hence we have a wide variety of products we can choose. So we narrow it down to either a burger (Zinger) or something from sakae sushi.

At last Teriyaki Chicken Ramen will be our product coz not only does it covers all the ingredients listed in this package but our group felt that exporting a japanese food is much more challenging compared to a burger. Furthermore, it's not as if America did not have its own burgers!

So the battle on the research of Teriyaki Chicken Ramen begins!

Categorize

Splitting the workload...

Meat - Apple

Egg - Jing Guo

Dairy - Hui Qi

Fruits & Vegetables - Fiona

Flour - Ming Hui

Fats & Oil - Rose

Knew...

Meat/ Poultry – Campylobacter Enterocolitis, Salmonellosis, Clostridium perfringens Food Infection, Staphylococcal Food Poisoning
Egg – Salmonellosis
Fruits & Veg – Over-ripening/ Heat exposure
Flour & flour base – Packaging pinholes/ Pest intrusion
Fats & Oils – Heat or Oxygen exposure

To find..

- Exact ingredients & process procedures
- Major pathogenic causes and the symptoms involved
- Singapore’s local authority; its system and specifications.
(AVA, Sale of Food Act, Singapore Standard Code of Practice)
- Overseas’ authority specifications in ensuring food safety (US FDA, NEA)

Food Safety:"How safe is the food on your plate?"

Everyone plays an important role in food safety; from farmers to food industry production-line workers to the cook at your very own home. It is essential to ensure the safety of food to prevent food-borne illness. Here are some tips on food safety!

Buying

• Check both production and/or expiry date
• Ensure that there is no default in packaging (eg: dented cans, unsealed)
• Appearence and quality of food should also be checked.
• In the industry, always check the product specification and Certificate of Analysis (CoA)

Storage

• Always follow the first-in-first-out(FIFO) rule
• Store food products at proper storage temperature
• Never store raw food product next to ready-to-eat food

In the industry

• Hazard Analysis Critical Control Point (HACCP) has been designed to ensure the safety of food in the food industry. It ensures food safety through biological, chemical and physical hazard identification and control in the production process.

Personal care

• Hands must ALWAYS be clean, washed or gloved when handling food
• Hairnet and/or face mask should be used
• Dont wear jewelleries or any body-accesories during the preparation of food product
• Dont be involved in the production of food if you are sick or feeling unwell

Work Area

• CLEAN
• Promptly wash any equipments after use

Production

• Food should not be left exposed for long
• Cook or prepare food at the right temperature and time

Remember, food safety is prevent microbiological, chemical and physical cross-contamination. Not only does the shelf-life of food greatly depend upon it, consumers too!

-know what you eat-